Cotton cultivar DP 444 BG/RR

ABSTRACT

A novel cotton cultivar, designated DP 444 BG/RR, is disclosed. The invention relates to the seeds of cotton cultivar DP 444 BG/RR, to the plants of cotton DP 444 BG/RR and to methods for producing a cotton plant produced by crossing the cultivar DP 444 BG/RR with itself or another cotton variety. The invention further relates to hybrid cotton seeds and plants produced by crossing the cultivar DP 444 BG/RR with another cotton cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a cotton (Gossypium) seed, a cottonplant, a cotton variety and a cotton hybrid. This invention furtherrelates to a method for producing cotton seed and plants.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm. Incotton, the important traits include higher fiber (lint) yield, earliermaturity, improved fiber quality, resistance to diseases and insects,resistance to drought and heat, and improved agronomic traits.

Pureline cultivars of cotton are commonly bred by hybridization of twoor more parents followed by selection. The complexity of inheritance,the breeding objectives and the available resources influence thebreeding method. Pedigree breeding, recurrent selection breeding andbackcross breeding are breeding methods commonly used in self pollinatedcrops such as cotton. These methods refer to the manner in whichbreeding pools or populations are made in order to combine desirabletraits from two or more cultivars or various broad-based sources. Theprocedures commonly used for selection of desirable individuals orpopulations of individuals are called mass selection, plant-to-rowselection and single seed descent or modified single seed descent. One,or a combination of these selection methods, can be used in thedevelopment of a cultivar from a breeding population.

Pedigree breeding is primarily used to combine favorable genes into atotally new cultivar that is different in many traits than either parentused in the original cross. It is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁ (filial generation 1).An F₂ population is produced by selfing F₁ plants. Selection ofdesirable individual plants may begin as early as the F₂ generationwherein maximum gene segregation occurs. Individual plant selection canoccur for one or more generations. Successively, seed from each selectedplant can be planted in individual, identified rows or hills, known asprogeny rows or progeny hills, to evaluate the line and to increase theseed quantity, or, to further select individual plants. Once a progenyrow or progeny hill is selected as having desirable traits it becomeswhat is known as a breeding line that is specifically identifiable fromother breeding lines that were derived from the same originalpopulation. At an advanced generation (i.e., F₅ or higher) seed ofindividual lines are evaluated in replicated testing. At an advancedstage the best lines or a mixture of phenotypically similar lines fromthe same original cross are tested for potential release as newcultivars.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep, et al. 1979; Fehr,1987).

The single seed descent procedure in the strict sense refers to plantinga segregating population, harvesting one seed from every plant, andcombining these seeds into a bulk which is planted the next generation.When the population has been advanced to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. Primary advantages of the seed descentprocedures are to delay selection until a high level of homozygosity(e.g., lack of gene segregation) is achieved in individual plants, andto move through these early generations quickly, usually through usingwinter nurseries.

The modified single seed descent procedures involve harvesting multipleseed (i.e., a single lock or a simple boll) from each plant in apopulation and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population.

Selection for desirable traits can occur at any segregating generation(F₂ and above). Selection pressure is exerted on a population by growingthe population in an environment where the desired trait is maximallyexpressed and the individuals or lines possessing the trait can beidentified. For instance, selection can occur for disease resistancewhen the plants or lines are grown in natural or artificially-induceddisease environments, and the breeder selects only those individualshaving little or no disease and are thus assumed to be resistant.

Promising advanced breeding lines are thoroughly tested and compared topopular cultivars in environments representative of the commercialtarget area(s) for three or more years. The best lines havingsuperiority over the popular cultivars are candidates to become newcommercial cultivars. Those lines still deficient in a few traits arediscarded or utilized as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from seven to twelve years from the time thefirst cross is made. Therefor, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior because, for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental lines and widely grown standardcultivars. For many traits a single observation is inconclusive, andreplicated observations over time and space are required to provide agood estimate of a line's genetic worth.

The goal of a commercial cotton breeding program is to develop new,unique and superior cotton cultivars. The breeder initially selects andcrosses two or more parental lines, followed by generation advancementand selection, thus producing many new genetic combinations. The breedercan theoretically generate billions of different genetic combinationsvia this procedure. The breeder has no direct control over which geneticcombinations will arise in the limited population size which is grown.Therefore, two breeders will never develop the same line having the sametraits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The lines whichare developed are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce, with any reasonable likelihood, thesame cultivar twice by using the exact same original parents and thesame selection techniques. This unpredictability results in theexpenditure of large amounts of research moneys to develop superior newcotton cultivars.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, and the grower, processor and consumer; forspecial advertising and marketing and commercial production practices,and new product utilization. The testing preceding the release of a newcultivar should take into consideration research and development costsas well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Cotton, Gossypium hirsutum, is an important and valuable field crop.Thus, a continuing goal of plant breeders is to develop stable, highyielding cotton cultivars that are agronomically sound. The reasons forthis goal are obviously to maximize the amount and quality of the fiberproduced on the land used and to supply fiber, oil and food for animalsand humans. To accomplish this goal, the cotton breeder must select anddevelop plants that have the traits that result in superior cultivars.

The development of new cotton cultivars requires the evaluation andselection of parents and the crossing of these parents. The lack ofpredictable success of a given cross requires that a breeder, in anygiven year, make several crosses with the same or different breedingobjectives.

The cotton flower is monecious in that the male and female structuresare in the same flower. The crossed or hybrid seed is produced by manualcrosses between selected parents. Floral buds of the parent that is tobe the female are emasculated prior to the opening of the flower bymanual removal of the male anthers. At flowering, the pollen fromflowers of the parent plants designated as male, are manually placed onthe stigma of the previous emasculated flower. Seed developed from thecross is known as first generation (F₁) hybrid seed. Planting of thisseed produces F₁ hybrid plants of which half their genetic component isfrom the female parent and half from the male parent. Segregation ofgenes begins at meiosis thus producing second generation (F₂) seed.Assuming multiple genetic differences between the original parents, eachF₂ seed has a unique combination of genes.

SUMMARY OF THE INVENTION

The present invention relates to a cotton seed, a cotton plant, a cottonvariety and a method for producing a cotton plant.

The present invention further relates to a method of producing cottonseeds and plants by crossing a plant of the instant invention withanother cotton plant.

This invention further relates to the seeds of cotton variety DP 444BG/RR, to the plants of cotton variety DP 444 BG/RR and to methods forproducing a cotton plant produced by crossing the cotton DP 444 BG/RRwith itself or another cotton line. Thus, any such methods using thecotton variety DP 444 BG/RR are part of this invention including:selfing, backcrosses, hybrid production, crosses to populations, and thelike.

In another aspect, the present invention provides for single traitconverted plants of DP 444 BG/RR. The single transferred trait maypreferably be a dominant or recessive allele. Preferably, the singletransferred trait will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced fiber quality, and industrialusage. The single trait may be a naturally occurring cotton gene or atransgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of cotton plant DP 444 BG/RR. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing cottonplant, and of regenerating plants having substantially the same genotypeas the foregoing cotton plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, orstems. Still further, the present invention provides cotton plantsregenerated from the tissue cultures of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Lint Yield. As used herein, the term “lint yield” is defined as themeasure of the quantity of fiber produced on a given unit of land.Presented below in pounds per acre.

Lint Percent. As used herein, the term “lint percent” is defined as thelint (fiber) fraction of seed cotton (lint and seed).

Gin Turnout. As used herein, the term “gin turnout” is defined as afraction of lint in a machine harvested sample of seed cotton (lint,seed, and trash).

Fiber Length (Len). As used herein, the term “fiber length” is definedas 2.5% span length in inches of fiber as measured by High VolumeInstrumentation (HVI).

Uniformity Ratio (Ur). As used herein, the term “uniformity ratio” isdefined as a measure of the relative length uniformity of a bundle offibers as measured by HVI.

Micronaire. As used herein, the term “micronaire” is defined as ameasure of the fineness of the fiber. Within a cotton cultivar,micronaire is also a measure of maturity. Micronaire differences aregoverned by changes in perimeter or in cell wall thickness, or bychanges in both. Within a variety, cotton perimeter is fairly constantand maturity will cause a change in micronaire. Consequently, micronairehas a high correlation with maturity within a variety of cotton.Maturity is the degree of development of cell wall thickness. Micronairemay not have a good correlation with maturity between varieties ofcotton having different fiber perimeter. Micronaire values range fromabout 2.0 to 6.0:

Below 2.9 Very fine Possible small perimeter but mature (good fiber), orlarge perimeter but immature (bad fiber). 2.9 to 3.7 Fine Variousdegrees of maturity and/or perimeter. 3.8 to 4.6 Average Average degreeof maturity and/or perimeter. 4.7 to 5.5 Coarse Usually fully developed(mature), but larger perimeter. 5.6+ Very coarse Fully developed,large-perimeter fiber.

Fiber Strength (T1). As used herein, the term “fiber strength” isdefined as the force required to break a bundle of fibers as measured ingrams per millitex on the HVI.

Fiber Elongation (E1). As used herein, the term “fiber elongation” isdefined as the measure of elasticity of a bundle of fibers as measuredby HVI.

Plant Height. As used herein, the term “plant height” is defined as theaverage height in inches or centimeters of a group of plants.

Stringout Rating (So). As used herein, the term “stringout rating” isdefined as a visual rating prior to harvest of the relative looseness ofthe seed cotton held in the boll structure on the plant.

Maturity Rating (Matur). As used herein, the term “maturity rating” isdefined as a visual rating near harvest on the amount of opened bolls onthe plant.

Vegetative Nodes. As used herein, the term “vegetative nodes” is definedas the number of nodes from the cotyledonary node to the first fruitingbranch on the main stem of the plant.

Seedweight (Sdwt). As used herein, the term “seedweight” is the weightof 100 seeds in grams.

Fallout (Fo). As used herein, the term “fallout” refers to the rating ofhow much cotton has fallen on the ground at harvest.

Lint Index. As used herein, the term “lint index” refers to the weightof lint per seed in milligrams.

Seed/boll. As used herein, the term “seed/boll” refers to the number ofseeds per boll.

Seedcotton/boll. As used herein, the term “seedcotton/boll” refers tothe weight of seedcotton per boll.

Lint/boll. As used herein, the term “lint/boll” is the weight of lintper boll.

Lint Percent. As used herein, the term “lint percent” refers to thepercentage of the seed cotton that is lint.

Fruiting Nodes. As used herein, the term “fruiting nodes” is defined asthe number of nodes on the main stem from which arise branches whichbear fruit or bolls.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted trait.

Single trait Converted (Conversion). Single trait converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single trait transferred intothe variety via the backcrossing technique or via genetic engineering.

Disease Resistance. As used herein the term “disease resistance” isdefined as the ability of plants to restrict the activities of aspecified pest, such as an insect, fungus, virus, or bacterial.

Disease Tolerance. As used herein the term “disease tolerance” isdefined as the ability of plants to endure a specified pest (such as aninsect, fungas, virus or bacteria) or an adverse environmental conditionand still performing and producing in spite of this disorder.

VRDP. As used herein the term “VRDP” is defined as the alleledesignation for the single dominant allele of the present inventionwhich confers virus resistance. VRDP designates “Virus ResistanceDeltapine”.

Variety Description Information

Species: Gossypium hirsutum L.

Areas of Adaptation: Eastern, Western, Central and Arizona

General:

Plant Habit Compact Foliage Sparse Stem Lodging Intermediate FruitingBranch Short Growth Intermediate Leaf Color Light green Boll ShapeLength greater than width Boll Breadth Broadest at base Maturity: Dateof 50% open bolls: September 16 Plant: No. of Nodes to 1st FruitingBranch 4.3 (Excluding cotyledonary node) Mature Plant Height 110 cm(from cotyledonary node to terminal) Leaf (Upper most, fully expandedleaf): Type Normal Pubescence Semi-hairy Nectaries Present StemPubescence: Intermediate Glands: Leaf Normal Stem Normal Calyx LobeNormal Flower: Petals Cream Pollen Cream Petal Spot Absent Seed: SeedIndex (g/100, fuzzy basis) 10.6 Lint Index (g lint/100 seeds) 4.36 Boll:Lint Percent - Picked 39.2% Number of Seeds per Boll 33.4 Grams SeedCotton per Boll 4.5 Number of Locules per Boll 4-5 Boll Type Open FiberProperties: Method HVI Length 1.15 inches Uniformity 84% Strength (T1)29.6 g/tex Elongation (E1) 11.7% Micronaire 4.0 Nematodes, Insects andPests: Pink Bollworm Resistant

This invention is also directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant, wherein the first or second cotton plant is the cotton plant fromthe line DP 444 BG/RR. Further, both the first and second parent cottonplants may be the cultivar DP 444 BG/RR (e.g., self-pollination).Therefore, any methods using the cultivar DP 444 BG/RR are part of thisinvention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using cultivar DP 444 BG/RR as a parentare within the scope of this invention. As used herein, the term “plant”includes plant cells, plant protoplasts, plant cells of tissue culturefrom which cotton plants can be regenerated, plant calli, plant clumps,and plant cells that are intact in plants or parts of plants, such aspollen, flowers, embryos, ovules, seeds, pods, leaves, stems, roots,anthers and the like. Thus, another aspect of this invention is toprovide for cells which upon growth and differentiation produce acultivar having essentially all of the physiological and morphologicalcharacteristics of DP 444 BG/RR.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, cotton is transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation, inMethods in Plant Molecular Biology & Biotechnology” in Glich, et al.,(Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors andGUS gene cassettes are available from Clone Tech Laboratories, Inc.,Palo Alto, Calif. while luciferase expression vectors and luciferasegene cassettes are available from Pro Mega Corp. (Madison, Wis.).General methods of culturing plant tissues are provided for example byMaki, et al., “Procedures for Introducing Foreign DNA into Plants” inMethods in Plant Molecular Biology & Biotechnology, Glich, et al., (Eds.pp. 67-88 CRC Press, 1993); and by Phillips, et al., “Cell-TissueCulture and In-Vitro Manipulation” in Corn & Corn Improvement, 3rdEdition; Sprague, et al., (Eds. pp. 345-387) American Society ofAgronomy Inc., 1988. Methods of introducing expression vectors intoplant tissue include the direct infection or co-cultivation of plantcells with Agrobacterium tumefaciens, Horsch et al., Science, 227:1229(1985). Descriptions of Agrobacterium vectors systems and methods forAgrobacterium-mediated gene transfer provided by Gruber, et al., supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice Agrobacterium-medicated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

The present invention contemplates a cotton plant regenerated from atissue culture of a variety (e.g., DP 444 BG/RR) or hybrid plant of thepresent invention. As is well known in the art, tissue culture of cottoncan be used for the in vitro regeneration of a cotton plant. Tissueculture of various tissues of cotton and regeneration of plantstherefrom is well known and widely published.

When the term cotton plant is used in the context of the presentinvention, this also includes any single trait conversions of thatvariety. The term single trait converted plant as used herein refers tothose cotton plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single trait transferred intothe variety. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to the recurrent parent. The parental cotton plant whichcontributes the trait for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental cotton plant to which the traitor traits from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a cotton plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a gene or genes of the recurrent varietyare modified or substituted with the desired gene(s) from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. Traits may or may not be transgenic, examplesof these traits include but are not limited to, male sterility,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility, enhanced fiber quality,industrial usage, yield stability and yield enhancement. These traitsare generally inherited through the nucleus.

The cultivar DP 444 BG/RR is similar to SG 501 BR. While similar, thereare numerous differences including: DP 444 BG/RR has a higher lintpercent, longer fiber and lower micronaire.

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed cotton plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the cotton plant(s).

Expression Vectors for Cotton Transformation: Marker Genes—Expressionvectors include at least one genetic marker, operably linked to aregulatory element (a promoter, for example) that allows transformedcells containing the marker to be either recovered by negativeselection, i.e., inhibiting growth of cells that do not contain theselectable marker gene, or by positive selection, i.e., screening forthe product encoded by the genetic marker. Many commonly used selectablemarker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) under the control of plantregulatory signals confers resistance to kanamycin. Fraley et al., Proc.Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990< Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS, β-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which intitiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in cotton. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in cotton. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in cotton or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in cotton.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in cotton.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in cotton. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993). Signal Sequences for TargetingProteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein then can be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a cotton plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encoddes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as nematodes. See e.g.,PCT Application WO96/30517; PCT Application WO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δendotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT application U.S.Pat. No. 93/06487, the contents of which are hereby incorporated byreference. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including theost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaid\cvirus, tobacco streak virus, potato virus X, potato virus Y, tobaccoetch virus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. C f.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivting gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, For Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II ).

Methods for Cotton Transformation—Numerous methods for planttransformation have been developed, including biological and physical,plant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

A. Agrobacterium-Mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of cotton target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular cotton line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Tissue Culture of Cotton—When the term cotton plant is used in thecontext of the present invention, this also includes any single geneconversions of that variety. The term single gene converted plant asused herein refers to those cotton plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thevariety. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent. The parentalcotton plant which contributes the gene for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental cotton plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the single gene of interest to be transferred. The resultingprogeny from this cross are then crossed again to the recurrent parentand the process is repeated until a cotton plant is obtained whereinessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of cottons andregeneration of plants therefrom is well know and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports(1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine-wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S.Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., the disclosuresof which are hereby incorporated herein in their entirety by reference.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce cotton plants having thephysiological and morphological characteristics of cotton variety DP 444BG/RR.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445,described certain techniques, the disclosures of which are incorporatedherein by reference.

This invention also is directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant wherein the first or second parent cotton plant is a cotton plantof the variety DP 444 BG/RR. Further, both first and second parentcotton plants can come from the cotton variety DP 444 BG/RR. Thus, anysuch methods using the cotton variety DP 444 BG/RR are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cotton variety DP444 BG/RR as a parent are within the scope of this invention, includingthose developed from varieties derived from cotton variety DP 444 BG/RR.Advantageously, the cotton variety could be used in crosses with other,different, cotton plants to produce first generation (F₁) cotton hybridseeds and plants with superior characteristics. The variety of theinvention can also be used for transformation where exogenous genes areintroduced and expressed by the variety of the invention. Geneticvariants created either through traditional breeding methods usingvariety DP 444 BG/RR or through transformation of DP 444 BG/RR by any ofa number of protocols known to those of skill in the art are intended tobe within the scope of this invention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which cotton plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, pods, leaves,roots, root tips, anthers, and the like.

TABLE 1 Multiple Location Rankings - 2001 Lint Yield Lint % Mic Len URT1 DP 444 BG/RR 1404 41.0 4.1 1.15 84 29.6 SG 501 B/RR 1357 38.9 4.81.11 84 30.2 DP 451 B/RR 1290 36.6 4.5 1.15 84 28.5 No. of tests inaverage 14 14 14 14 14 14

Deposit Information

A deposit of the Delta and Pine Land Company cotton cultivar DP 444BG/RR disclosed above and recited in the appended claims has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassa, Va. 20110. The date of deposit was Jul. 22, 2004.The deposit of 2,500 seeds was taken from the same deposit maintained byDelta and Pine Land Company since prior to the filing date of thisapplication. All restrictions upon the deposit have been removed, andthe deposit is intended to meet all of the requirements of 37 C.F.R.§1,801-1.809. The ATCC accession number is PTA-6126. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

1. Seed of a cotton variety designated DP 444 BG/RR wherein a sample ofseed was deposited under ATCC Accession No. PTA-6126.
 2. A cotton plant,or a part thereof, of variety DP 444 BG/RR, seed of said variety havingbeen deposited under ATCC Accession No. PTA-6126.
 3. Pollen of the plantof claim
 2. 4. An ovule of the plant of claim
 2. 5. A tissue culture ofregenerable cells from the plant of claim
 2. 6. A tissue cultureaccording to claim 5, wherein said cell or a protoplast of the tissueculture is derived from a tissue selected from the group consisting ofleaves, pollen, embryos, cotyledon, hypocotyl, meristematic cells,roots, root tips, anthers, flowers, seeds, stems and pods.
 7. The cottonplant regenerated from the tissue culture of claim 5, wherein theregenerated plant has all of the morphological and physiologicalcharacteristics of cotton cultivar DP 444 BG/RR and wherein a sample ofseed was deposited under ATCC Accession No. PTA-6126.
 8. A method forproducing a hybrid cotton seed comprising crossing a first parent cottonplant with a second parent cotton plant and harvesting the resultanthybrid cotton seed, wherein said first parent cotton plant or saidsecond parent cotton plant is the cotton plant of claim
 2. 9. A methodfor producing a transgenic cotton plant or a transgenic part thereof,said method comprising transforming the cotton plant of claim 2 or apart thereof with a transgene operably linked to a regulatory element,wherein said transgene confers a trait selected from the groupconsisting of herbicide resistance, insect resistance and diseaseresistance.
 10. A transgenic cotton plant or transgenic part thereof,produced by the method of claim
 9. 11. A cotton plant according to claim10, wherein said herbicide resistance is to glyphosate, glufosinate, asulfonylurea or imidazolinone herbicide, or a protoporphyrinogen oxidaseinhibitor.
 12. A method for producing a cotton plant that contains inits genetic material a transgene, comprising crossing the cotton plantof claim 2 with a cotton plant containing a transgene, so that thegenetic material of the progeny that result from the cross contains atransgene operably linked to a regulatory element and wherein saidtransgene confers a trait selected from the group consisting ofherbicide resistance, insect resistance and disease resistance.